Customer Service

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

An infectious extract of Nematocida-infected worms is prepared and cryopreserved as follows:

Harvest infected nematodes when most worms are filled with spores. Using a balanced saline buffer solution such as ATCC medium 1323, M9 buffer, or a PBS solution, wash nematode worms into suspension and transfer to a 15-ml centrifuge tube.

Centrifuge at 1500 x g for 30 sec, rinse 3 times with distilled water, let sit for 1 hr, then rinse again 2 times with distilled water.

Filter the worm lysate is through a 5 µm filter (Millipore) attached to a syringe in order to eliminate undisrupted C. elegans eggs, larvae, and other debris. (Filter becomes saturated after passing ~100 µl packed nematodes; use additional filters as necessary.)

Place the vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion. At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately -1°C/min.)

Store in either the vapor or liquid phase of a nitrogen refrigerator.

To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 1 minute. Do not agitate the ampule. Do not leave ampule in water bath after thawed.

When completely thawed, dilute the spore preparation by addition of 0.25 to 0.5 ml of a balanced saline buffer solution such as ATCC medium 1323, M9 buffer, or a PBS solution.

Infect C. elegans nematodes by adding the dilute spore preparation onto an agar plate containing an established C. elegans culture. Seal the plate with parafilm and incubate upright at 25°C. Follow the protocol for maintenance in-vivo.

Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.